Polymerase chain reaction, hereinafter PCR, is a method of amplifying a DNA target sequence, i.e. a method of producing a large number of copies of a given sequence of DNA, in a relatively short period of time. A PCR reaction solution therefore includes a DNA template having the target sequence, a heat-resistant DNA polymerase, typically Taq or Pfu, a pair of primers, i.e. a pair of short, single-stranded sequences complimentary to the 3′ end of the target sequence, and nucleotides to form the sequence copies.
Once the solution is mixed in a PCR tube, the tube is placed in a thermal cycler and exposed to a series of temperature cycles which enable the target sequence to be denatured, each primer to anneal (i.e. bind) to the relevant strand of the target sequence and the nucleotides to bind the primers to form a new strand of DNA complementary to the denatured target sequence in the 5′ to 3′ direction. The thermal cycles described above are repeated a number of times, usually up to 30.
As PCR allows the production of a large number of copies of target sequences in hours, it is widely used in cloning, genetic engineering, sequencing, functional analysis of genes, molecular detection and diagnosis of hereditary or infectious diseases and identification of genetic fingerprints.
PCR cycles can be subdivided into an exponential phase during which each thermal cycle duplicates the number of target sequences and a plateau phase during which inhibitors of the polymerase reaction found in the amplified sample, nucleotide limitation, accumulation of pyrophosphate molecules, and self-annealing of the accumulating product results in amplification of the target sequence ceasing to occur. If the reaction is halted during the exponential phase, the quantity of starting target sequence can be determined. This is useful, for example in forensic applications in which it is necessary to determine the quantity of starting material. However, if the reaction is carried out until it reaches the plateau phase, it is not possible to quantify the amount of starting material. Accordingly, a newer method based on PCR and called real time PCR or quantitative real time polymerase chain reaction (qPCR) was developed to enable simultaneous amplification, detection and quantification of the target sequence.
Real time PCR is largely similar to traditional PCR in terms of reagents with the addition of intercalating non-specific double-stranded DNA binding fluorescent dyes, fluorescently labeled nucleotides or phosphorus-32 labeled nucleotides. Accordingly, a qPCR thermal cycler combines a thermal system with an optical system capable of detecting fluorescence or radiation and, in addition, software to control the apparatus, collect and analyse data.
Regardless of whether standard PCR or qPCR is used, fluorescence spectroscopy is used in combination with PCR techniques in order to detect specific sequences or to quantify the amount of DNA present in a sample. In fluorescence spectroscopy, there are two aspects that require validation. Firstly, the instrument itself is validated (fluorescence intensity and spectral correction) by comparison to a Certified Reference Material (CRM), also known as Standard Reference Material (SRM). Secondly, analyte detection measurements are also validated by reference to a SRM. As most analyte detection is carried out in a solution, validation of analyte detection measurements is usually performed by comparing the measurement to a solution containing a fluorescent dye.
Metrology is a science which is concerned with measurement; specifically, it includes experimental and theoretical determinations in any field of technology. The international vocabulary of metrology is maintained by the International Organisation for Standardisation (ISO) and is currently in its third revision (VIM 3).
One of the most important concepts in metrology is metrological traceability, which is defined in the ISO/IEC Guide 99:2007 (International vocabulary of metrology—Basic and general concepts and associated terms (VIM)) as the “property of a measurement result whereby the result can be related to a reference through a documented unbroken chain of calibrations, each contributing to the measurement uncertainty.” In other words, traceability is the property of the result of a measurement whereby it can be related to references, usually national or international standards, through an unbroken chain of comparisons all having stated uncertainties.
In many countries, national standards for weights and measures are maintained by National Metrology Institutes (NMIs) which provide the highest level of standards for the calibration or measurement traceability infrastructure in that country. For example, in the UK, one NMI is the National Physical Laboratory (NPL); in the US, the NMI is called National Institute of Standards and Technology (NIST); in Germany, one NMI is the Physikalisch-Technische Bundesanstalt (PTB); and, in Canada, the NMI is the NRC Institute for National Measurement Standards (NRC).
Typically, traceability is achieved by calibration which establishes the relation between the result shown in a measuring instrument and the value of a measured standard. Thus, calibration to a traceable standard can be used to determine whether an instrument is precise and accurate and it can also be used to determine whether the instrument has a bias.
There is currently no standard method used to validate thermal cyclers having means for measuring fluorescence of a sample; this is highly undesirable because it means that fluorescence measurements obtained in thermal cyclers are not traceable. Further, use of thermal cyclers in regulated environments has increased dramatically in recent times, accordingly, the requirement to qualify these systems has also increased.
The present invention seeks to provide means for validating a thermal cycler. Further, the present invention aims to provide a method for validating a thermal cycler.